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The vertebrate heart tube extends by progressive addition of epithelial second heart field (SHF) progenitor cells from the dorsal pericardial wall. The interplay between epithelial mechanics and genetic mechanisms during SHF deployment is unknown. Here, we present a quantitative single-cell morphometric analysis of SHF cells during heart tube extension, including force inference analysis of epithelial stress. Joint spatial Principal Component Analysis reveals that cell orientation and stress direction are the main parameters defining apical cell morphology and distinguishes cells adjacent to the arterial and venous poles. Cell shape and mechanical forces display a dynamic relationship during heart tube formation. Moreover, while the T-box transcription factor Tbx1 is necessary for cell orientation towards the arterial pole, activation of Tbx5 in the posterior SHF correlates with the establishment of epithelial stress and SHF deletion of Tbx5 relaxes the progenitor epithelium. Integrating findings from cell-scale feature patterning and mechanical stress provides new insights into cardiac morphogenesis., (© 2024. The Author(s).)

Congenital heart defects (CHD) affect 1 in 100 live births and result from defects in cardiac development. Growth of the early heart tube occurs by the progressive addition of second heart field (SHF) progenitor cells to the cardiac poles. The SHF gives rise to ventricular septal, right ventricular and outflow tract myocardium at the arterial pole, and atrial, including atrial septal myocardium, at the venous pole. SHF deployment creates the template for subsequent cardiac septation and has been implicated in cardiac looping and in orchestrating outflow tract development with neural crest cells. Genetic or environmental perturbation of SHF deployment thus underlies a spectrum of common forms of CHD affecting conotruncal and septal morphogenesis. Here we review the major properties of SHF cells as well as recent insights into the developmental programs that drive normal cardiac progenitor cell addition and the origins of CHD.

The major events of cardiac development, including early heart formation, chamber morphogenesis and septation, and conduction system and coronary artery development, are briefly reviewed together with a short introduction to the animal species commonly used to study heart development and model congenital heart defects (CHDs)., (© 2024. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

Development of the outflow tract of the heart requires specification, proliferation and deployment of a progenitor cell population from the second heart field to generate the myocardium at the arterial pole of the heart. Disruption of these processes leads to lethal defects in rotation and septation of the outflow tract. We previously showed that Fibroblast Growth Factor 8 (FGF8) directs a signaling cascade in the second heart field that regulates critical aspects of OFT morphogenesis. Here we show that in addition to the survival and proliferation cues previously described, FGF8 provides instructive and patterning information to OFT myocardial cells and their progenitors that prevents their aberrant differentiation along a working myocardial program., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Organogenesis requires the orchestrated development of multiple cell lineages that converge, interact, and specialize to generate coherent functional structures, exemplified by transformation of the cardiac crescent into a four-chambered heart. Cardiomyocytes originate from the first and second heart fields, which make different regional contributions to the definitive heart. In this review, a series of recent single-cell transcriptomic analyses, together with genetic tracing experiments, are discussed, providing a detailed panorama of the cardiac progenitor cell landscape. These studies reveal that first heart field cells originate in a juxtacardiac field adjacent to extraembryonic mesoderm and contribute to the ventrolateral side of the cardiac primordium. In contrast, second heart field cells are deployed dorsomedially from a multilineage-primed progenitor population via arterial and venous pole pathways. Refining our knowledge of the origin and developmental trajectories of cells that build the heart is essential to address outstanding challenges in cardiac biology and disease., Competing Interests: Declaration of interests The author declares no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Branchiomeric muscles of the head and neck originate in a population of cranial mesoderm termed cardiopharyngeal mesoderm that also contains progenitor cells contributing to growth of the embryonic heart. Retrospective lineage analysis has shown that branchiomeric muscles share a clonal origin with parts of the heart, indicating the presence of common heart and head muscle progenitor cells in the early embryo. Genetic lineage tracing and functional studies in the mouse, as well as in Ciona and zebrafish, together with recent experiments using single cell transcriptomics and multipotent stem cells, have provided further support for the existence of bipotent head and heart muscle progenitor cells. Current challenges concern defining where and when such common progenitor cells exist in mammalian embryos and how alternative myogenic derivatives emerge in cardiopharyngeal mesoderm. Addressing these questions will provide insights into mechanisms of cell fate acquisition and the evolution of vertebrate musculature, as well as clinical insights into the origins of muscle restricted myopathies and congenital defects affecting craniofacial and cardiac development., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Epithelial cardiac progenitor cells of the second heart field (SHF) contribute to growth of the vertebrate heart tube by progressive addition of cells from the dorsal pericardial wall to the cardiac poles. Perturbation of SHF development, including defects in apicobasal or planar polarity, results in shortening of the heart tube and a spectrum of congenital heart defects. Here, we provide detailed protocols for fixed section and wholemount immunofluorescence and live imaging approaches to studying the epithelial properties of cardiac progenitors in the dorsal pericardial wall during mouse heart development. Whole-embryo culture and electroporation methods are also presented, allowing for pharmacological and genetic perturbation of SHF development, as well as image analysis approaches to quantify cell features across the progenitor cell epithelium. These protocols are broadly applicable to the study of epithelia in the early embryo., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

The ventricular conduction system coordinates heartbeats by rapid propagation of electrical activity through the Purkinje fiber (PF) network. PFs share common progenitors with contractile cardiomyocytes, yet the mechanisms of segregation and network morphogenesis are poorly understood. Here, we apply genetic fate mapping and temporal clonal analysis to identify murine cardiomyocytes committed to the PF lineage as early as E7.5. We find that a polyclonal PF network emerges by progressive recruitment of conductive precursors to this scaffold from a pool of bipotent progenitors. At late fetal stages, the segregation of conductive cells increases during a phase of rapid recruitment to build the definitive PF network through a non-cell autonomous mechanism. We also show that PF differentiation is impaired in Nkx2-5 haploinsufficient embryos leading to failure to extend the scaffold. In particular, late fetal recruitment fails, resulting in PF hypoplasia and persistence of bipotent progenitors. Our results identify how transcription factor dosage regulates cell fate divergence during distinct phases of PF network morphogenesis.

The mammalian heart contains multiple cell types that appear progressively during embryonic development. Advance in determining cardiac lineage diversification has often been limited by the unreliability of genetic tracers. Here we combine clonal analysis, genetic lineage tracing, tissue transplantation, and mutant characterization to investigate the lineage relationships between epicardium, arterial mesothelial cells (AMCs), and the coronary vasculature. We report a contribution of the second heart field (SHF) to a vasculogenic niche composed of AMCs and sub-mesothelial cells at the base of the pulmonary artery. Sub-mesothelial cells from this niche differentiate into lymphatic endothelial cells and, in close association with AMC-derived cells, contribute to and are essential for the development of ventral cardiac lymphatics. In addition, regionalized epicardial/mesothelial retinoic acid signaling regulates lymphangiogenesis, contributing to the niche properties. These results uncover a SHF vasculogenic contribution to coronary lymphatic development through a local niche at the base of the great arteries., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Tbx1, the major candidate gene for DiGeorge or 22q11.2 deletion syndrome, is required for efficient incorporation of cardiac progenitors of the second heart field (SHF) into the heart. However, the mechanisms by which TBX1 regulates this process are still unclear. Here, we have used two independent models, mouse embryos and cultured cells, to define the role of TBX1 in establishing morphological and dynamic characteristics of SHF in the mouse. We found that loss of TBX1 impairs extracellular matrix (ECM)-integrin-focal adhesion (FA) signaling in both models. Mosaic analysis in embryos suggested that this function is non-cell autonomous, and, in cultured cells, loss of TBX1 impairs cell migration and FAs. Additionally, we found that ECM-mediated integrin signaling is disrupted upon loss of TBX1. Finally, we show that interfering with the ECM-integrin-FA axis between E8.5 and E9.5 in mouse embryos, corresponding to the time window within which TBX1 is required in the SHF, causes outflow tract dysmorphogenesis. Our results demonstrate that TBX1 is required to maintain the integrity of ECM-cell interactions in the SHF and that this interaction is critical for cardiac outflow tract development. More broadly, our data identifies a novel TBX1 downstream pathway as an important player in SHF tissue architecture and cardiac morphogenesis., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

In vertebrates, multipotent progenitors located in the pharyngeal mesoderm form cardiomyocytes and branchiomeric head muscles, but the dynamic gene expression programmes and mechanisms underlying cardiopharyngeal multipotency and heart versus head muscle fate choices remain elusive. Here, we used single-cell genomics in the simple chordate model Ciona to reconstruct developmental trajectories forming first and second heart lineages and pharyngeal muscle precursors and characterize the molecular underpinnings of cardiopharyngeal fate choices. We show that FGF-MAPK signalling maintains multipotency and promotes the pharyngeal muscle fate, whereas signal termination permits the deployment of a pan-cardiac programme, shared by the first and second heart lineages, to define heart identity. In the second heart lineage, a Tbx1/10-Dach pathway actively suppresses the first heart lineage programme, conditioning later cell diversity in the beating heart. Finally, cross-species comparisons between Ciona and the mouse evoke the deep evolutionary origins of cardiopharyngeal networks in chordates.

The vertebrate heart tube forms from epithelial progenitor cells in the early embryo and subsequently elongates by progressive addition of second heart field (SHF) progenitor cells from adjacent splanchnic mesoderm. Failure to maximally elongate the heart results in a spectrum of morphological defects affecting the cardiac poles, including outflow tract alignment and atrioventricular septal defects, among the most common congenital birth anomalies. SHF cells constitute an atypical apicobasally polarized epithelium with dynamic basal filopodia, located in the dorsal wall of the pericardial cavity. Recent studies have highlighted the importance of epithelial architecture and cell adhesion in the SHF, particularly for signaling events that control the progenitor cell niche during heart tube elongation. The 22q11.2 deletion syndrome gene Tbx1 regulates progenitor cell status through modulating cell shape and filopodial activity and is required for SHF contributions to both cardiac poles. Noncanonical Wnt signaling and planar cell polarity pathway genes control epithelial polarity in the dorsal pericardial wall, as progenitor cells differentiate in a transition zone at the arterial pole. Defects in these pathways lead to outflow tract shortening. Moreover, new biomechanical models of heart tube elongation have been proposed based on analysis of tissue-wide forces driving epithelial morphogenesis in the SHF, including regional cell intercalation, cell cohesion, and epithelial tension. Regulation of the epithelial properties of SHF cells is thus emerging as a key step during heart tube elongation, adding a new facet to our understanding of the mechanisms underlying both heart morphogenesis and congenital heart defects., (© 2017 American Heart Association, Inc.)

Extension of the vertebrate heart tube is driven by progressive addition of second heart field (SHF) progenitor cells to the poles of the heart. Defects in this process cause a spectrum of congenital anomalies. SHF cells form an epithelial layer in splanchnic mesoderm in the dorsal wall of the pericardial cavity. Here we report oriented cell elongation, polarized actomyosin distribution and nuclear YAP/TAZ in a proliferative centre in the posterior dorsal pericardial wall during heart tube extension. These parameters are indicative of mechanical stress, further supported by analysis of cell shape changes in wound assays. Time course and mutant analysis identifies SHF deployment as a source of epithelial tension. Moreover, cell division and oriented growth in the dorsal pericardial wall align with the axis of cell elongation, suggesting that epithelial tension in turn contributes to heart tube extension. Our results implicate tissue-level forces in the regulation of heart tube extension.

The transcription factors Mesp1 and Mesp2 have essential roles in the migration and specification of multipotent progenitor cells at the onset of cardiogenesis. Chiapparo et al. (2016. J. Cell Biol http://dx.doi.org/10.1083/jcb.201505082) identify common Mesp functions in fate specification and Mesp1-specific targets controlling the speed and direction of progenitor cell migration., (© 2016 Kelly.)

Background: Coronary artery (CA) stems connect the ventricular coronary tree with the aorta. Defects in proximal CA patterning are a cause of sudden cardiac death. In mice lacking Tbx1, common arterial trunk is associated with an abnormal trajectory of the proximal left CA. Here we investigate CA stem development in wild-type and Tbx1 null embryos., Results: Genetic lineage tracing reveals that limited outgrowth of aortic endothelium contributes to proximal CA stems. Immunohistochemistry and fluorescent tracer injections identify a periarterial vascular plexus present at the onset of CA stem development. Transplantation experiments in avian embryos indicate that the periarterial plexus originates in mesenchyme distal to the outflow tract. Tbx1 is required for the patterning but not timing of CA stem development and a Tbx1 reporter allele is expressed in myocardium adjacent to the left but not right CA stem. This expression domain is maintained in Sema3c(-/-) hearts with a common arterial trunk and leftward positioned CA. Ectopic myocardial differentiation is observed on the left side of the Tbx1(-/-) common arterial trunk., Conclusions: A periarterial plexus bridges limited outgrowth of the aortic endothelium with the ventricular plexus during CA stem development. Molecular differences associated with left and right CA stems provide new insights into the etiology of CA patterning defects., (© 2016 Wiley Periodicals, Inc.)

Congenital coronary artery anomalies are of major significance in clinical cardiology and cardiac surgery due to their association with myocardial ischaemia and sudden death. Such anomalies are detectable by imaging modalities and, according to various definitions, their prevalence ranges from 0.21 to 5.79%. This consensus document from the Development, Anatomy and Pathology Working Group of the European Society of Cardiology aims to provide: (i) a definition of normality that refers to essential anatomical and embryological features of coronary vessels, based on the integrated analysis of studies of normal and abnormal coronary embryogenesis and pathophysiology; (ii) an animal model-based systematic survey of the molecular and cellular mechanisms that regulate coronary blood vessel development; (iii) an organization of the wide spectrum of coronary artery anomalies, according to a comprehensive anatomical and embryological classification scheme; (iv) current knowledge of the pathophysiological mechanisms underlying symptoms and signs of coronary artery anomalies, with diagnostic and therapeutic implications. This document identifies the mosaic-like embryonic development of the coronary vascular system, as coronary cell types differentiate from multiple cell sources through an intricate network of molecular signals and haemodynamic cues, as the necessary framework for understanding the complex spectrum of coronary artery anomalies observed in human patients., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2016. For permissions please email: journals.permissions@oup.com.)

Differential inductive signaling during asymmetric division of progenitor cells specifies the heart lineage in Ciona intestinalis. In this issue of Developmental Cell, Cota and Davidson (2015) show that differential induction is mediated by FGF receptor regionalization, resulting from asymmetric cell-matrix adhesion and reduced mitotic turnover of polarized Caveolin-rich membrane domains., (Copyright © 2015 Elsevier Inc. All rights reserved.)

It has been more than 30 years since the publication of the new head hypothesis, which proposed that the vertebrate head is an evolutionary novelty resulting from the emergence of neural crest and cranial placodes. Neural crest generates the skull and associated connective tissues, whereas placodes produce sensory organs. However, neither crest nor placodes produce head muscles, which are a crucial component of the complex vertebrate head. We discuss emerging evidence for a surprising link between the evolution of head muscles and chambered hearts - both systems arise from a common pool of mesoderm progenitor cells within the cardiopharyngeal field of vertebrate embryos. We consider the origin of this field in non-vertebrate chordates and its evolution in vertebrates.

Outflow tract (OFT) malformation accounts for ∼30% of human congenital heart defects and manifests frequently in TBX1 haplo-insufficiency associated DiGeorge (22q11.2 deletion) syndrome. OFT myocardium originates from second heart field (SHF) progenitors in the pharyngeal and splanchnic mesoderm (SpM), but how these progenitors are deployed to the OFT is unclear. We find that SHF progenitors in the SpM gradually gain epithelial character and are deployed to the OFT as a cohesive sheet. Wnt5a, a non-canonical Wnt, is expressed specifically in the caudal SpM and may regulate oriented cell intercalation to incorporate SHF progenitors into an epithelial-like sheet, thereby generating the pushing force to deploy SHF cells rostrally into the OFT. Using enhancer trap and Cre transgenes, our lineage tracing experiments show that in Wnt5a null mice, SHF progenitors are trapped in the SpM and fail to be deployed to the OFT efficiently, resulting in a reduction in the inferior OFT myocardial wall and its derivative, subpulmonary myocardium. Concomitantly, the superior OFT and subaortic myocardium are expanded. Finally, in chick embryos, blocking the Wnt5a function in the caudal SpM perturbs polarized elongation of SHF progenitors, and compromises their deployment to the OFT. Collectively, our results highlight a critical role for Wnt5a in deploying SHF progenitors from the SpM to the OFT. Given that Wnt5a is a putative transcriptional target of Tbx1, and the similar reduction of subpulmonary myocardium in Tbx1 mutant mice, our results suggest that perturbing Wnt5a-mediated SHF deployment may be an important pathogenic mechanism contributing to OFT malformations in DiGeorge syndrome., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Neck muscles constitute a transition zone between somite-derived skeletal muscles of the trunk and limbs, and muscles of the head, which derive from cranial mesoderm. The trapezius and sternocleidomastoid neck muscles are formed from progenitor cells that have expressed markers of cranial pharyngeal mesoderm, whereas other muscles in the neck arise from Pax3-expressing cells in the somites. Mef2c-AHF-Cre genetic tracing experiments and Tbx1 mutant analysis show that nonsomitic neck muscles share a gene regulatory network with cardiac progenitor cells in pharyngeal mesoderm of the second heart field (SHF) and branchial arch-derived head muscles. Retrospective clonal analysis shows that this group of neck muscles includes laryngeal muscles and a component of the splenius muscle, of mixed somitic and nonsomitic origin. We demonstrate that the trapezius muscle group is clonally related to myocardium at the venous pole of the heart, which derives from the posterior SHF. The left clonal sublineage includes myocardium of the pulmonary trunk at the arterial pole of the heart. Although muscles derived from the first and second branchial arches also share a clonal relationship with different SHF-derived parts of the heart, neck muscles are clonally distinct from these muscles and define a third clonal population of common skeletal and cardiac muscle progenitor cells within cardiopharyngeal mesoderm. By linking neck muscle and heart development, our findings highlight the importance of cardiopharyngeal mesoderm in the evolution of the vertebrate heart and neck and in the pathophysiology of human congenital disease.

Aims: Cardiomyocyte proliferation gradually declines during embryogenesis resulting in severely limited regenerative capacities in the adult heart. Understanding the developmental processes controlling cardiomyocyte proliferation may thus identify new therapeutic targets to modulate the cell-cycle activity of cardiomyocytes in the adult heart. This study aims to determine the mechanism by which fibroblast growth factor 10 (FGF10) controls foetal cardiomyocyte proliferation and to test the hypothesis that FGF10 promotes the proliferative capacity of adult cardiomyocytes., Methods and Results: Analysis of Fgf10(-/-) hearts and primary cardiomyocyte cultures reveals that altered ventricular morphology is associated with impaired proliferation of right but not left-ventricular myocytes. Decreased FOXO3 phosphorylation associated with up-regulated p27(kip) (1) levels was observed specifically in the right ventricle of Fgf10(-/-) hearts. In addition, cell-type-specific expression analysis revealed that Fgf10 and its receptor, Fgfr2b, are expressed in cardiomyocytes and not cardiac fibroblasts, consistent with a cell-type autonomous role of FGF10 in regulating regional specific myocyte proliferation in the foetal heart. Furthermore, we demonstrate that in vivo overexpression of Fgf10 in adult mice promotes cardiomyocyte but not cardiac fibroblast cell-cycle re-entry., Conclusion: FGF10 regulates regional cardiomyocyte proliferation in the foetal heart through a FOXO3/p27(kip1) pathway. In addition, FGF10 triggers cell-cycle re-entry of adult cardiomyocytes and is thus a potential target for cardiac repair., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2014. For permissions please email: journals.permissions@oup.com.)

Elongation of the vertebrate heart occurs by progressive addition of second heart field (SHF) cardiac progenitor cells from pharyngeal mesoderm to the poles of the heart tube. The importance of these cells in the etiology of congenital heart defects has led to extensive research into the regulation of SHF deployment by signaling pathways and transcription factors. However, the basic cellular features of these progenitor cells, including epithelial polarity, cell shape and cell dynamics, remain poorly characterized. Here, using immunofluorescence, live imaging and embryo culture, we demonstrate that SHF cells constitute an atypical, apicobasally polarized epithelium in the dorsal pericardial wall, characterized by apical monocilia and dynamic actin-rich basal filopodia. We identify the 22q11.2 deletion syndrome gene Tbx1, required in the SHF for outflow tract development, as a regulator of the epithelial properties of SHF cells. Cell shape changes in mutant embryos include increased circularity, a reduced basolateral membrane domain and impaired filopodial activity, and are associated with elevated aPKCζ levels. Activation of aPKCζ in embryo culture similarly impairs filopodia activity and phenocopies proliferative defects and ectopic differentiation observed in the SHF of Tbx1 null embryos. Our results reveal that epithelial and progenitor cell status are coupled in the SHF, identifying control of cell shape as a regulatory step in heart tube elongation and outflow tract morphogenesis., (© 2014. Published by The Company of Biologists Ltd.)

Rationale: Cardiac progenitor cells from the second heart field (SHF) contribute to rapid growth of the embryonic heart, giving rise to right ventricular and outflow tract (OFT) myocardium at the arterial pole of the heart, and atrial myocardium at the venous pole. Recent clonal analysis and cell-tracing experiments indicate that a common progenitor pool in the posterior region of the SHF gives rise to both OFT and atrial myocytes. The mechanisms regulating deployment of this progenitor pool remain unknown., Objective: To evaluate the role of TBX1, the major gene implicated in congenital heart defects in 22q11.2 deletion syndrome patients, in posterior SHF development., Methods and Results: Using transcriptome analysis, genetic tracing, and fluorescent dye-labeling experiments, we show that Tbx1-dependent OFT myocardium originates in Hox-expressing cells in the posterior SHF. In Tbx1 null embryos, OFT progenitor cells fail to segregate from this progenitor cell pool, leading to failure to expand the dorsal pericardial wall and altered positioning of the cardiac poles. Unexpectedly, addition of SHF cells to the venous pole of the heart is also impaired, resulting in abnormal development of the dorsal mesenchymal protrusion, and partially penetrant atrioventricular septal defects, including ostium primum defects., Conclusions: Tbx1 is required for inflow as well as OFT morphogenesis by regulating the segregation and deployment of progenitor cells in the posterior SHF. Our results provide new insights into the pathogenesis of congenital heart defects and 22q11.2 deletion syndrome phenotypes., (© 2014 American Heart Association, Inc.)

In this review, we focus on two important steps in the formation of the embryonic heart: (i) the progressive addition of late differentiating progenitor cells from the second heart field that drives heart tube extension during looping morphogenesis, and (ii) the emergence of patterned proliferation within the embryonic myocardium that generates distinct cardiac chambers. During the transition between these steps, the major site of proliferation switches from progenitor cells outside the early heart to proliferation within the embryonic myocardium. The second heart field and ballooning morphogenesis concepts have major repercussions on our understanding of human heart development and disease. In particular, they provide a framework to dissect the origin of congenital heart defects and the regulation of myocardial proliferation and differentiation of relevance for cardiac repair., (Copyright © 2014 Cold Spring Harbor Laboratory Press; all rights reserved.)

Congenital heart defects affect at least 0.8% of newborn children and are a major cause of lethality prior to birth. Malformations of the arterial pole are particularly frequent. The myocardium at the base of the pulmonary trunk and aorta and the arterial tree associated with these great arteries are derived from splanchnic mesoderm of the second heart field (SHF), an important source of cardiac progenitor cells. These cells are controlled by a gene regulatory network that includes Fgf8, Fgf10 and Tbx1. Prdm1 encodes a transcriptional repressor that we show is also expressed in the SHF. In mouse embryos, mutation of Prdm1 affects branchial arch development and leads to persistent truncus arteriosus (PTA), indicative of neural crest dysfunction. Using conditional mutants, we show that this is not due to a direct function of Prdm1 in neural crest cells. Mutation of Prdm1 in the SHF does not result in PTA, but leads to arterial pole defects, characterized by mis-alignment or reduction of the aorta and pulmonary trunk, and abnormalities in the arterial tree, defects that are preceded by a reduction in outflow tract size and loss of caudal pharyngeal arch arteries. These defects are associated with a reduction in proliferation of progenitor cells in the SHF. We have investigated genetic interactions with Fgf8 and Tbx1, and show that on a Tbx1 heterozygote background, conditional Prdm1 mutants have more pronounced arterial pole defects, now including PTA. Our results identify PRDM1 as a potential modifier of phenotypic severity in TBX1 haploinsufficient DiGeorge syndrome patients., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

At the end of the first week of mouse gestation, cardiomyocyte differentiation initiates in the cardiac crescent to give rise to the linear heart tube. The heart tube subsequently elongates by addition of cardiac progenitor cells from adjacent pharyngeal mesoderm to the growing arterial and venous poles. These progenitor cells, termed the second heart field, originate in splanchnic mesoderm medial to cells of the cardiac crescent and are patterned into anterior and posterior domains adjacent to the arterial and venous poles of the heart, respectively. Perturbation of second heart field cell deployment results in a spectrum of congenital heart anomalies including conotruncal and atrial septal defects seen in human patients. Here, we briefly review current knowledge of how the properties of second heart field cells are controlled by a network of transcriptional regulators and intercellular signaling pathways. Focus will be on 1) the regulation of cardiac progenitor cell proliferation in pharyngeal mesoderm, 2) the control of progressive progenitor cell differentiation and 3) the patterning of cardiac progenitor cells in the dorsal pericardial wall. Coordination of these three processes in the early embryo drives progressive heart tube elongation during cardiac morphogenesis. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Cardiac Pathways of Differentiation, Metabolism and Contraction., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Organogenesis of the vertebrate heart involves a complex sequence of events initiating with specification and differentiation of myocardial and endocardial cells in anterior lateral mesoderm shortly after gastrulation, followed by formation and rightward looping of the early heart tube. During looping, the heart tube elongates by addition of second heart field progenitor cells from adjacent pharyngeal mesoderm at the arterial and venous poles. Progressive differentiation is controlled by intercellular signaling events between pharyngeal mesoderm, foregut endoderm, and neural crest-derived mesenchyme. Regulated patterns of myocardial gene expression and proliferation within the embryonic heart drive morphogenesis of atrial and ventricular chambers, while cardiac cushions, precursors of the definitive valves, form in the atrioventricular and outflow regions. In amniotes, separate systemic and pulmonary circulatory systems arise by septation and remodeling events that divide the atria and ventricles into left and right chambers. Cardiac neural crest cells play a key role in dividing the arterial pole of the heart into the ascending aorta and pulmonary trunk. During the remodeling phase the definitive cardiac conduction system, that coordinates the heartbeat, is established. In addition, the epicardium, critical for regulated ventricular growth and development of the coronary vasculature, spreads over the surface of the heart as an epithelium from which cells invade the myocardium to give rise to diverse cell types including fibroblasts and smooth muscle cells. Cardiogenesis thus involves highly coordinated development of multiple cell types and insight into the different lineage contributions and molecular regulation of each of these steps is expanding rapidly. WIREs Dev Biol 2013, 2:17-29. doi: 10.1002/wdev.68 For further resources related to this article, please visit the WIREs website., (Copyright © 2012 Wiley Periodicals, Inc.)

During cardiogenesis, Fibroblast Growth Factor (Fgf10) is expressed in the anterior second heart field. Together with Fibroblast growth factor 8 (Fgf8), Fgf10 promotes the proliferation of these cardiac progenitor cells that form the arterial pole of the heart. We have identified a 1.7-kb region in the first intron of Fgf10 that is necessary and sufficient to direct transgene expression in this cardiac context. The 1.7-kb sequence is directly controlled by T-box transcription factor 1 (Tbx1) in anterior second heart field cells that contribute to the outflow tract. It also responds to both NK2 transcription factor related, locus 5 (Nkx2-5) and ISL1 transcription factor, LIM/homeodomain (Islet1), acting through overlapping sites. Mutation of these sites reduces transgene expression in the anterior second heart field where the Fgf10 regulatory element is activated by Islet1 via direct binding in vivo. Analysis of the response to Nkx2-5 loss- and Isl1 gain-of-function genetic backgrounds indicates that the observed up-regulation of its activity in Nkx2-5 mutant hearts, reflecting that of Fgf10, is due to the absence of Nkx2-5 repression and to up-regulation of Isl1, normally repressed in the myocardium by Nkx2-5. ChIP experiments show strong binding of Nkx2-5 in differentiated myocardium. Molecular and genetic analysis of the Fgf10 cardiac element therefore reveals how key cardiac transcription factors orchestrate gene expression in the anterior second heart field and how genes, such as Fgf10, normally expressed in the progenitor cell population, are repressed when these cells enter the heart and differentiate into myocardium. Our findings provide a paradigm for transcriptional mechanisms that underlie the changes in regulatory networks during the transition from progenitor state to that of the differentiated tissue.

During cardiac looping the heart tube elongates by addition of progenitor cells from adjacent pharyngeal mesoderm to the arterial and venous poles. This cell population, termed the second heart field, was first identified ten years ago and many studies in the intervening decade have refined our understanding of how heart tube elongation takes place and identified signaling pathways that regulate proliferation and differentiation during progressive contribution of second heart field cells to the embryonic heart. It has also become apparent that defective second heart field development results in common congenital heart anomalies affecting both the conotruncal region and venous pole of the heart, including atrial and atrioventricular septal defects. In this review we focus on a series of recent papers that have identified new regulators of second heart field development, in particular the retinoic acid signaling pathway and HOX, SIX and EYA transcription factors. We also discuss new findings concerning the regulation of fibroblast growth factor signaling during second heart field deployment and studies that have implicated FGF10 and FGF3 in outflow tract development in addition to FGF8. Second heart field derived parts of the heart share common progenitor cells in pharyngeal mesoderm with craniofacial skeletal muscles and recent findings from xenopus, zebrafish and the protochordate Ciona intestinalis provide insights into the evolution of the second heart field during vertebrate radiation., (Copyright © 2012 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.)

Ten years ago, a population of cardiac progenitor cells was identified in pharyngeal mesoderm that gives rise to a major part of the amniote heart. These multipotent progenitor cells, termed the second heart field (SHF), contribute progressively to the poles of the elongating heart tube during looping morphogenesis, giving rise to myocardium, smooth muscle, and endothelial cells. Research into the mechanisms of SHF development has contributed significantly to our understanding of the properties of cardiac progenitor cells and the origins of congenital heart defects. Here recent data concerning the regulation, clinically relevant subpopulations, evolution and lineage relationships of the SHF are reviewed. Proliferation and differentiation of SHF cells are controlled by multiple intercellular signaling pathways and a transcriptional regulatory network that is beginning to be elucidated. Perturbation of SHF development results in common forms of congenital heart defects and particular progenitor cell subpopulations are highly relevant clinically, including cells giving rise to myocardium at the base of the pulmonary trunk and the interatrial septum. A SHF has recently been identified in amphibian, fish, and agnathan embryos, highlighting the important contribution of these cells to the evolution of the vertebrate heart. Finally, SHF-derived parts of the heart share a lineage relationship with craniofacial skeletal muscles revealing that these progenitor cells belong to a broad cardiocraniofacial field of pharyngeal mesoderm. Investigation of the mechanisms underlying the dynamic process of SHF deployment is likely to yield further insights into cardiac development and pathology., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Head muscle progenitors in pharyngeal mesoderm are present in close proximity to cells of the second heart field and show overlapping patterns of gene expression. However, it is not clear whether a single progenitor cell gives rise to both heart and head muscles. We now show that this is the case, using a retrospective clonal analysis in which an nlaacZ sequence, converted to functional nlacZ after a rare intragenic recombination event, is targeted to the alpha(c)-actin gene, expressed in all developing skeletal and cardiac muscle. We distinguish two branchiomeric head muscle lineages, which segregate early, both of which also contribute to myocardium. The first gives rise to the temporalis and masseter muscles, which derive from the first branchial arch, and also to the extraocular muscles, thus demonstrating a contribution from paraxial as well as prechordal mesoderm to this anterior muscle group. Unexpectedly, this first lineage also contributes to myocardium of the right ventricle. The second lineage gives rise to muscles of facial expression, which derive from mesoderm of the second branchial arch. It also contributes to outflow tract myocardium at the base of the arteries. Further sublineages distinguish myocardium at the base of the aorta or pulmonary trunk, with a clonal relationship to right or left head muscles, respectively. We thus establish a lineage tree, which we correlate with genetic regulation, and demonstrate a clonal relationship linking groups of head muscles to different parts of the heart, reflecting the posterior movement of the arterial pole during pharyngeal morphogenesis.

22q11 deletion syndrome (22q11DS) is characterised by aberrant development of the pharyngeal apparatus and the heart with haploinsufficiency of the transcription factor TBX1 being considered the major underlying cause of the disease. Tbx1 mutations in mouse phenocopy the disorder. In order to identify the transcriptional dysregulation in Tbx1-expressing lineages we optimised fluorescent-activated cell sorting of beta-galactosidase expressing cells (FACS-Gal) to compare the expression profile of Df1/Tbx1(lacZ) (effectively Tbx1 null) and Tbx1 heterozygous cells isolated from mouse embryos. Hes1, a major effector of Notch signalling, was identified as downregulated in Tbx1(-)(/)(-) mutants. Hes1 mutant mice exhibited a partially penetrant range of 22q11DS-like defects including pharyngeal arch artery (PAA), outflow tract, craniofacial and thymic abnormalities. Similar to Tbx1 mice, conditional mutagenesis revealed that Hes1 expression in embryonic pharyngeal ectoderm contributes to thymus and pharyngeal arch artery development. These results suggest that Hes1 acts downstream of Tbx1 in the morphogenesis of pharyngeal-derived structures., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

Background: Rapid growth of the embryonic heart occurs by addition of progenitor cells of the second heart field to the poles of the elongating heart tube. Failure or perturbation of this process leads to congenital heart defects. In order to provide further insight into second heart field development we characterized the insertion site of a transgene expressed in the second heart field and outflow tract as the result of an integration site position effect., Results: Here we show that the integration site of the A17-Myf5-nlacZ-T55 transgene lies upstream of Hes1, encoding a basic helix-loop-helix containing transcriptional repressor required for the maintenance of diverse progenitor cell populations during embryonic development. Transgene expression in a subset of Hes1 expression sites, including the CNS, pharyngeal epithelia, pericardium, limb bud and lung endoderm suggests that Hes1 is the endogenous target of regulatory elements trapped by the transgene. Hes1 is expressed in pharyngeal endoderm and mesoderm including the second heart field. Analysis of Hes1 mutant hearts at embryonic day 15.5 reveals outflow tract alignment defects including ventricular septal defects and overriding aorta. At earlier developmental stages, Hes1 mutant embryos display defects in second heart field proliferation, a reduction in cardiac neural crest cells and failure to completely extend the outflow tract., Conclusions: Hes1 is expressed in cardiac progenitor cells in the early embryo and is required for development of the arterial pole of the heart.

Insight into the mechanisms underlying congenital heart defects and the use of stem cells for cardiac repair are major research goals in cardiovascular biology. In the early embryo, progenitor cells in pharyngeal mesoderm contribute to the rapid growth of the heart tube during looping morphogenesis. These progenitor cells constitute the second heart field (SHF) and were first identified in 2001. Direct or indirect perturbation of SHF addition to the heart results in congenital heart defects, including arterial pole alignment defects. Over the last 3 years, a number of studies have identified key intercellular signaling pathways that control the proliferation and deployment of SHF progenitor cells. Here, we review data concerning Wnt, fibroblast growth factor, bone morphogenetic protein, Hedgehog, and retinoic acid signaling that have begun to identify the ligand sources and responding cell types controlling SHF development. These studies have revealed the importance of signals from pharyngeal mesoderm itself, as well as critical inputs from adjacent pharyngeal epithelia and neural crest cells. Proliferation is emerging as a central checkpoint in the regulation of SHF development. Together, these studies contribute to defining the niche of cardiac progenitor cells in the early embryo, and we discuss the implications of these findings for the regulation of resident stem cell populations in the fetal and postnatal heart. Characterization of signals that maintain, expand, and regulate the differentiation of cardiac progenitor cells is essential for understanding both the etiology of congenital heart defects and the biomedical application of stem cell populations for cardiac repair.

Conotruncal and ventricular septal congenital heart anomalies result from defects in formation and division of the embryonic outflow tract. Cardiac remodeling during outflow tract and ventricular septation converts the tubular embryonic heart into a parallel circulatory system with an independent left ventricular outlet and right ventricular inlet. Tbx3 encodes a T-box-containing transcription factor expressed in the developing conduction system of the heart. Mutations in TBX3 cause ulnar-mammary syndrome. Here we show that mice lacking Tbx3 develop severe outflow tract defects, including connection of both the aorta and pulmonary trunk with the right ventricle, in addition to aortic arch artery anomalies and abnormal communication between the right atrium and left ventricle. Alignment defects are preceded by a delay in caudal displacement of the arterial pole of the heart during aortic arch artery formation. Embryonic anterior-posterior patterning and cardiac chamber development are unaffected in Tbx3 mutant embryos. However, the contribution of second heart field derived progenitor cells to the arterial pole of the heart is impaired. Tbx3 is expressed in pharyngeal epithelia and neural crest cells in the pharyngeal region, suggesting an indirect role in second heart field deployment. Loss of Tbx3 affects multiple signaling pathways regulating second heart field proliferation and outflow tract morphogenesis, including fibroblast growth factor signaling, leading to a failure of normal heart tube extension and consequent atrioventricular and ventriculoarterial alignment defects.

Outflow tract myocardium in the mouse heart is derived from the anterior heart field, a subdomain of the second heart field. We have recently characterized a transgene (y96-Myf5-nlacZ-16), which is expressed in the inferior wall of the outflow tract and then predominantly in myocardium at the base of the pulmonary trunk. Transgene A17-Myf5-nlacZ-T55 is expressed in the developing heart in a complementary pattern to y96-Myf5-nlacZ-16, in the superior wall of the outflow tract at E10.5 and in myocardium at the base of the aorta at E14.5. At E9.5, the two transgenes are transcribed in different subdomains of the anterior heart field. A clonal analysis of cardiomyocytes in the outflow tract, at E10.5 and E14.5, provides insight into the behaviour of myocardial cells and their progenitors. At E14.5, most clones are located at the base of either the pulmonary trunk or the aorta, indicating that these derive from distinct myocardial domains. At E10.5, clones are observed in subdomains of the outflow tract. The distribution of small clones indicates proliferative differences, whereas regionalization of large clones, that derive from an early myocardial progenitor cell, reflect coherent cell growth in the heart field as well as in the myocardium. Our results suggest that myocardial differences at the base of the great arteries are prefigured in distinct progenitor cell populations in the anterior heart field, with important implications for understanding the etiology of congenital heart defects affecting the arterial pole of the heart.

Advances in our understanding of cardiac development have fuelled research into cellular approaches to myocardial repair of the damaged heart. In this collection of reviews we present recent advances into the basic mechanisms of heart development and the resident and non-resident progenitor cell populations that are currently being investigated as potential mediators of cardiac repair. Together these reviews illustrate that despite our current knowledge about how the heart is constructed, caution and much more research in this exciting field is essential. The current momentum to evaluate the potential for cardiac repair will in turn accelerate research into fundamental aspects of myocardial biology.

Tbx1, the major gene underlying del22q11.2 or DiGeorge syndrome in humans, is required for normal development and septation of the cardiac outflow tract. The fibroblast growth factor 10 gene (Fgf10) and an Fgf10 enhancer trap transgene are expressed in outflow tract myocardial progenitor cells of the anterior heart field. To visualize outflow tract development in the absence of Tbx1, we have analyzed the expression profile of the Fgf10 enhancer trap transgene during outflow tract development in Tbx1(-/-) embryos. Transgene expression confirms hypoplasia of the distal outflow tract in the absence of Tbx1, and altered expression in pharyngeal mesoderm reveals loss of specific bilateral subpopulations of outflow tract progenitor cells and disruption of the posterior boundary of the anterior heart field. Our results support the conclusion that Tbx1 controls deployment of Fgf10-expressing progenitor cells during heart tube extension. Furthermore, although normal Fgf10 levels are dependent on Tbx1, loss of Fgf10 alleles does not significantly modify the cardiac phenotype of Tbx1 heterozygous or homozygous mutant embryos.

Objective: Myocardial progenitor cells expressing Fgf10 give rise to the outflow tract and right ventricle of the mammalian heart. In order to define the role of fibroblast growth factor (FGF) signaling in this process we investigated whether Fgf10 or the major Fgf10 receptor Fgfr2-IIIb are required for normal heart development., Methods: The cardiac phenotype of Fgf10 and Fgfr2-IIIb mutant mice was analysed by histology, scanning electron microscopy and gene and transgene expression studies., Results: Outflow tract formation from Fgf10 expressing progenitor cells occurs normally in Fgf10 mutant embryos and in the majority of Fgfr2-IIIb mutant embryos; a proportion of Fgfr2-IIIb mutant embryos, however, display outflow tract and right ventricular hypoplasia. The predominant cardiac defects in Fgfr2-IIIb mutant embryos are ventricular septal defects associated with overriding aorta or double outlet right ventricle. In addition, loss of Fgfr2-IIIb is associated with ventricular anomalies including a thin myocardial wall, abnormal trabeculation and muscular ventricular septal defects. In contrast, Fgf10 is required to correctly position the heart in the thoracic cavity but not for outflow tract septation. Both Fgf10 and Fgfr2-IIIb mutant embryos lack pulmonary arteries and veins., Conclusions: Fgfr2-IIIb and Fgf10 mutant mice have distinct roles during cardiac morphogenesis, although neither gene is essential for outflow tract elongation from Fgf10 expressing progenitor cells. Fgfr2-IIIb and Fgf10 mutant mice provide new models for common components of congenital heart disease.

Mammalian heart development involves complex morphogenetic events which lead to the formation of fully separated left and right atrial and ventricular chambers from a tubular heart. Separation of left and right ventricular chambers is dependent on a single structure, the interventricular septum (IVS), which has both muscular and mesenchymal components. Little is known about the morphogenetic events that lead to the formation of the muscular component of the IVS. We have analyzed two transgenic mouse lines that display complementary nlacZ reporter gene expression patterns in the embryonic ventricles: the Mlc1v-nlacZ-24 transgene is expressed in right ventricular myocardium and the Mlc3f-nlacZ-2 transgene in left ventricular myocardium. Detailed analysis of these transgene expression patterns during IVS formation reveals a symmetric left and right myocardial identity within the developing IVS between embryonic days 9.5 and 11.5. From embryonic day 12.5 onwards, myocytes with a left ventricular identity dominate the IVS, particularly in its dorsal aspect. The T-box transcription factor encoding gene, Tbx18, is expressed in the left ventricle and left side of the developing IVS, providing additional support for the presence of left and right ventricular identities within the IVS. Analysis of clonally related cardiomyocyte clusters confirms that both left and right ventricular myocardial cell populations contribute to the forming IVS, in similar domains to those defined by the Mlc-nlacZ transgenes. Examination of the orientation as well as the distribution of labeled cells in clusters provides new insights into the morphogenesis of the septum.

Congenital heart defects frequently involve a failure of outflow tract (OFT) formation during development. We analyzed the remodeling of the OFT, using the y96-Myf5-nlacZ-16 transgene, which marks a subpopulation of myocardial cells of the pulmonary trunk. Expression analyses of reporter transcript and protein suggest that the myocardial wall of the OFT rotates before and during the formation of the great arteries. Rotational movement was confirmed by Di-I injection experiments with cultured embryos. We subsequently examined the expression of the transgene in mouse models for OFT defects. In hearts with persistent truncus arteriosus (PTA), double outlet right ventricle (DORV), or transposition of the great arteries, rotation of the myocardial wall of the OFT is arrested or fails to initiate. This is observed in Splotch (Pax3) mutants with PTA or DORV and may be a result of defects in neural crest migration, known to affect OFT septation. However, in Pitx2deltac mutant embryos, where cardiac neural crest cells are present in the heart, PTA and DORV are again associated with a rotation defect. This is also seen in Pitx2deltac mutants, which have transposition of the great arteries. Because Pitx2c is involved in left-right signaling, these results suggest that embryonic laterality affects rotation of the myocardial wall during OFT maturation. We propose that failure of normal rotation of OFT myocardium may underlie major forms of congenital heart disease.

In 2001, three research groups described a previously unrecognized population of progenitor cells in pharyngeal mesoderm that gives rise to myocardium at the arterial pole of the heart. In the last 4 years, the major importance of the cellular contribution of pharyngeal mesoderm to normal and pathologic heart development has become apparent. Lineage-tracing experiments have defined the extent to which pharyngeal progenitor cells colonize the heart, revealing a contribution to venous, as well as arterial, pole myocardium; in addition, major molecular inroads have been made into understanding gene regulation in pharyngeal myocardial progenitor cells, implicating forkhead, Gata, LIM homeodomain, MEF2, SMAD, and T-box transcription factors. The key role of the anterior heart field during normal heart development is underscored by the demonstration that both direct and indirect perturbation of myocardial progenitor cells in pharyngeal mesoderm result in congenital heart disease.

Tbx2 is a member of the T-box transcription factor gene family, and is expressed in a variety of tissues and organs during embryogenesis. In the developing heart, Tbx2 is expressed in the outflow tract, inner curvature, atrioventricular canal and inflow tract, corresponding to a myocardial zone that is excluded from chamber differentiation at 9.5 days post coitus (dpc). We have used targeted mutagenesis in mice to investigate Tbx2 function. Mice heterozygous for a Tbx2 null mutation appear normal but homozygous embryos reveal a crucial role for Tbx2 during cardiac development. Morphological defects are observed in development of the atrioventricular canal and septation of the outflow tract. Molecular analysis reveals that Tbx2 is required to repress chamber differentiation in the atrioventricular canal at 9.5 dpc. Analysis of homozygous mutants also highlights a role for Tbx2 during hindlimb digit development. Despite evidence that TBX2 negatively regulates the cell cycle control genes Cdkn2a, Cdkn2b and Cdkn1a in cultured cells, there is no evidence that loss of Tbx2 function during mouse development results in increased levels of p19(ARF), p16(INK4a), p15(INK4b) or p21 expression in vivo, nor is there evidence for a genetic interaction between Tbx2 and p53.

When and how cells form and pattern the myocardium is a central issue for heart morphogenesis. Many genes are differentially expressed and function in subsets of myocardial cells. However, the lineage relationships between these cells remain poorly understood. To examine this, we have adopted a retrospective approach in the mouse embryo, based on the use of the laacZ reporter gene, targeted to the alpha-cardiac actin locus. This clonal analysis demonstrates the existence of two lineages that segregate early from a common precursor. The primitive left ventricle and the presumptive outflow tract are derived exclusively from a single lineage. Unexpectedly, all other regions of the heart, including the primitive atria, are colonized by both lineages. These results are not consistent with the prespecification of the cardiac tube as a segmented structure. They are discussed in the context of different heart fields and of the evolution of the heart.

Key molecules which regulate the formation of the heart have been identified; however, the mechanism of cardiac morphogenesis remains poorly understood at the cellular level. We have adopted a genetic approach, which permits retrospective clonal analysis of myocardial cells in the mouse embryo, based on the targeting of an nlaacZ reporter to the alpha-cardiac actin gene. A rare intragenic recombination event leads to a clone of beta-galactosidase-positive myocardial cells. Analysis of clones at different developmental stages demonstrates that myocardial cells and their precursors follow a proliferative mode of growth, rather than a stem cell mode, with an initial dispersive phase, followed by coherent cell growth. Clusters of cells are dispersed along the venous-arterial axis of the heart tube. Coherent growth is oriented locally, with a main axis, which corresponds to the elongation of the cluster, and rows of cells, which form secondary axes. The angle between the primary and secondary axes varies, indicating independent events of growth orientation. At later stages, as the ventricular wall thickens, wedge shaped clusters traverse the wall and contain rows of cells at a progressive angle to each other. The cellular organisation of the myocardium appears to prefigure myofibre architecture. We discuss how the characteristics of myocardial cell growth, which we describe, underlie the formation of the heart tube and its subsequent regionalised expansion.

Studies of vertebrate heart development have identified key genes and signalling molecules involved in the formation of a myocardial tube from paired heart-forming fields in splanchnic mesoderm. The posterior region of the paired heart-forming fields subsequently contributes myocardial precursor cells to the inflow region or venous pole of the heart. Recently, a population of myocardial precursor cells in chick and mouse embryos has been identified in pharyngeal mesoderm anterior to the early heart tube. This anterior heart-forming field gives rise to myocardium of the outflow region or arterial pole of the heart. The amniote heart is therefore derived from two myocardial precursor cell populations, which appear to be regulated by distinct genetic programmes. Discovery of the anterior heart-forming field has important implications for the interpretation of cardiac defects in mouse mutants and for the study of human congenital heart disease.